Overexpression, Purification, and Characterization of the Purple Protein: pGEM-gbr22 Introduction & Objective:
Having the ability to create and isolate a concentrated amount of protein in solution is an essential process in recreating protein-ligand interactions in vitro. To begin, scientists identify a protein they wish to study and obtain a cDNA sequence that is responsible for expressing it [1]. The cDNA is cloned into an expression vector and a fusion tag of choice is appended to the sequence [2]. The vector is expressed in an organism, such as E. coli, and allowed to replicate. The protein is then solubilized and purified through a variety of methods involving filters and buffers that exploit the attached tag [3]. The resulting elution can be tested for protein concentration and characterized using a gel electrophoresis to determine purity and molecular weight. Further tests such as an enzyme assay can be performed on the protein to determine compounds that could function as inhibitors. This lab aims to use the methods mentioned above in order to obtain a relatively concentrated and pure elution of pGEM-gbr22, a protein found in coral with a distinctive purple color. If methods are followed properly the protein elution should be purple and its characterization should indicate a heavy concentration of protein at 25.79 kDa. Good introduction
Materials & Methods:
Part 1: Bacterial Protein Expression
Using sterile technique 25μL of BL21(DE3) E. coli (New England Biolabs, Ipswich, MA) was placed into two transformation tubes. One of the colonies was transformed with a pGEM-gbr22 plasmid using a heat shock method, and SOC media was added to both tubes. Both solutions were plated on an LB/Ampicillin plate (Figure 1, Figure 2); a swab of a laboratory keyboard and mouse was plated on a standard LB/Agar plate (Figure 3); all three were incubated overnight at 37° C. Ampicillin stock and a single colony of transformed bacteria were added to two tubes of LB in a sterile culture tube. Colonies were grown in a shaking incubator at for 8 hours at 37° C. Fresh LB, Ampicillin stock, and starter culture from one tube was added to a sterile Erlenmeyer flask. Colonies were grown in a shaking incubator overnight at 37° C. Once the culture was purple and turbid (Figure 4) the bacteria was centrifuged in a conical tube. The resulting pellet (Figure5) was suspended in 1X PBS and Lysozyme, then frozen at -20°C.
Part 2: Protein Purification
The suspension was thawed, and Cyanase was added. We used Benzonase in lab. The sample was centrifuged and filtered through a .22mm syringe PES filter. Ni-NTA resin/buffer was added to the suspension and allowed to settle. The solution was poured into a Bio-Rad chromatography column and allowed to flow through. The resin was washed with 20mM imidazole in 1X PBS. The protein was eluted through by adding a solution of 250mM Imidazole in 1X PBS (Figure6); this was repeated to create a second elution (Figure 7).
Absorbance of Elution 1 was recorded at λ=280nm and λ=574nm using a Thermo Fisher Scientific Inc. NanoDrop ND-1000 Spectrophotometer; n=2 (Figure 8). Write as: NanoDrop ND-100 Spectrophotometer (ThermoFisher, Waltham, MA) Part 3: Protein Characterization
Samples 1-6 (collected during expression and characterization) were prepared by adding loading buffer to create a 1X solution. Samples were denatured by placing them into a heat block at 95°C for 5 minutes and centrifuging. Samples and a Thermo Scientific Protein Ladder were loaded into a gel cassette. The gel was run in a mini-PROTEAN tank, then rinsed and stained using Imperial protein stain. It was then given time to destain in NanoPure overnight, before being vacuum dried (Figure 9).
Results:
RPM and temperature?
Missing a picture of the PAGE ladder from Thermo Scientific website
Discussion:
During expression, transformation was verified with the help of the ampicillin plates. The transformed bacteria grew successfully, indicating that the gbr22 gene containing ampicillin resistance was present in the organism. The unchanged E. coli served as a positive control: the lack of bacterial growth was an indicator that the untransformed strain had no natural ampicillin resistance. Lysozyme was added to the pellet suspension in order to break down the cell wall of the E. coli expressing gbr22. Improper sterile technique may have resulted in growth of other organisms and thus an impure final pellet.
During purification, Cyanase was added to the thawed pellet suspension to degrade all forms of DNA and RNA from the mixture. The HIS tag system works by attaching histidine residues to the C-terminus of the protein. These residues bind to nickel that can be immobilized on the Ni-NTA column matrix. The wash buffer is used to remove proteins that are only loosely bound to the resin, then the desired protein can be released from the Ni-NTA by adding the elution buffer containing a higher concentration of imidazole, which competes with the histidine residues for metal binding.
After purification was complete, the average protein concentration was calculated using Beer’s Law (A=ebc). A concentration of .205mg/mLwas detected at λ=280nm, and .082mg/mL at λ=574nm. There was a protein yield of .409mg at λ=574nm, and 1.02mg at λ=280.
As seen in Figure 9, six samples were run through the characterization gel. Sample 1: E. coli overexpressing pGEM-gbr22 collected after the overexpression was complete. Sample 2: lysed and Cyanase-treated E. coli at the beginning of purification. Sample 3: flow-through of supernatant flushed with Ni-NTA resin/buffer mix. Sample 4: Wash fraction of remaining proteins with 20mM Imidazole. Sample 5: first elution, using 250mM imidazole. Sample 5: second elution, using 250mM Imidazole.
Using the gel run during characterization, the molecular weight of gbr22 in Sample 5 was estimated to be ~25 kDa. This was consistent with its known molecular weight of 25.79 kDa. Apart from gbr22, Sample 5 also had a very faint band of unknown protein at ~13 kDa, indicating an overall protein purity of ~95%. The heavy banding in Samples 3 and 4 showed that the buffer and wash were successful in removing impurities from supernatant Sample 2. Faint banding in Elution 2 indicated that Elution 1 was successful in outcompeting Ni-NTA. Large bands at ~13 kDa in Samples 1, 2, and 3 showed that some impurity was present in solution. This may have been introduced because of poor lab technique, or more likely overexpressed by the bacteria during growth phase. Poor loading of the gel led to bleeding of bands, and background stain could have been avoided by further washing of the gel.
Conclusion:
The methods used to overexpress, purify, and characterize a protein were addressed throughout this lab. E. coli BL21(DE3) was transformed with the genetic sequence for protein pGEM-gbr22 and made to overexpress the purple protein. Through the use of physical filters, chemical tags, and various buffers, the protein was purified and assessed for concentration level. In characterization, various samples of genetic material were run through a gel to conclusively verify that the protein being detected was actually gbr22. Being able to apply these same techniques in experiments involving proteins of interest can allow for experiments such as enzyme assays or other tests to test enzyme inhibition.
References:
[1] Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46.
[3] Young, C. L.; Britton, Z. T.; Robinson, A. S., Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnol J 2012, 7 (5), 620-34.
Overexpression, Purification, and Characterization of the Purple Protein: pGEM-gbr22
Introduction & Objective:
Having the ability to create and isolate a concentrated amount of protein in solution is an essential process in recreating protein-ligand interactions in vitro. To begin, scientists identify a protein they wish to study and obtain a cDNA sequence that is responsible for expressing it [1]. The cDNA is cloned into an expression vector and a fusion tag of choice is appended to the sequence [2]. The vector is expressed in an organism, such as E. coli, and allowed to replicate. The protein is then solubilized and purified through a variety of methods involving filters and buffers that exploit the attached tag [3]. The resulting elution can be tested for protein concentration and characterized using a gel electrophoresis to determine purity and molecular weight. Further tests such as an enzyme assay can be performed on the protein to determine compounds that could function as inhibitors. This lab aims to use the methods mentioned above in order to obtain a relatively concentrated and pure elution of pGEM-gbr22, a protein found in coral with a distinctive purple color. If methods are followed properly the protein elution should be purple and its characterization should indicate a heavy concentration of protein at 25.79 kDa. Good introduction
Materials & Methods:
Part 1: Bacterial Protein Expression
Using sterile technique 25μL of BL21(DE3) E. coli (New England Biolabs, Ipswich, MA) was placed into two transformation tubes. One of the colonies was transformed with a pGEM-gbr22 plasmid using a heat shock method, and SOC media was added to both tubes. Both solutions were plated on an LB/Ampicillin plate (Figure 1, Figure 2); a swab of a laboratory keyboard and mouse was plated on a standard LB/Agar plate (Figure 3); all three were incubated overnight at 37° C. Ampicillin stock and a single colony of transformed bacteria were added to two tubes of LB in a sterile culture tube. Colonies were grown in a shaking incubator at for 8 hours at 37° C. Fresh LB, Ampicillin stock, and starter culture from one tube was added to a sterile Erlenmeyer flask. Colonies were grown in a shaking incubator overnight at 37° C. Once the culture was purple and turbid (Figure 4) the bacteria was centrifuged in a conical tube. The resulting pellet (Figure 5) was suspended in 1X PBS and Lysozyme, then frozen at -20°C.
Part 2: Protein Purification
The suspension was thawed, and Cyanase was added. We used Benzonase in lab. The sample was centrifuged and filtered through a .22mm syringe PES filter. Ni-NTA resin/buffer was added to the suspension and allowed to settle. The solution was poured into a Bio-Rad chromatography column and allowed to flow through. The resin was washed with 20mM imidazole in 1X PBS. The protein was eluted through by adding a solution of 250mM Imidazole in 1X PBS (Figure 6); this was repeated to create a second elution (Figure 7).
Absorbance of Elution 1 was recorded at λ=280nm and λ=574nm using a Thermo Fisher Scientific Inc. NanoDrop ND-1000 Spectrophotometer; n=2 (Figure 8).
Write as: NanoDrop ND-100 Spectrophotometer (ThermoFisher, Waltham, MA)
Part 3: Protein Characterization
Samples 1-6 (collected during expression and characterization) were prepared by adding loading buffer to create a 1X solution. Samples were denatured by placing them into a heat block at 95°C for 5 minutes and centrifuging. Samples and a Thermo Scientific Protein Ladder were loaded into a gel cassette. The gel was run in a mini-PROTEAN tank, then rinsed and stained using Imperial protein stain. It was then given time to destain in NanoPure overnight, before being vacuum dried (Figure 9).
Results:
Missing a picture of the PAGE ladder from Thermo Scientific website
Discussion:
During expression, transformation was verified with the help of the ampicillin plates. The transformed bacteria grew successfully, indicating that the gbr22 gene containing ampicillin resistance was present in the organism. The unchanged E. coli served as a positive control: the lack of bacterial growth was an indicator that the untransformed strain had no natural ampicillin resistance. Lysozyme was added to the pellet suspension in order to break down the cell wall of the E. coli expressing gbr22. Improper sterile technique may have resulted in growth of other organisms and thus an impure final pellet.
During purification, Cyanase was added to the thawed pellet suspension to degrade all forms of DNA and RNA from the mixture. The HIS tag system works by attaching histidine residues to the C-terminus of the protein. These residues bind to nickel that can be immobilized on the Ni-NTA column matrix. The wash buffer is used to remove proteins that are only loosely bound to the resin, then the desired protein can be released from the Ni-NTA by adding the elution buffer containing a higher concentration of imidazole, which competes with the histidine residues for metal binding.
After purification was complete, the average protein concentration was calculated using Beer’s Law (A=ebc). A concentration of .205mg/mLwas detected at λ=280nm, and .082mg/mL at λ=574nm. There was a protein yield of .409mg at λ=574nm, and 1.02mg at λ=280.
As seen in Figure 9, six samples were run through the characterization gel. Sample 1: E. coli overexpressing pGEM-gbr22 collected after the overexpression was complete. Sample 2: lysed and Cyanase-treated E. coli at the beginning of purification. Sample 3: flow-through of supernatant flushed with Ni-NTA resin/buffer mix. Sample 4: Wash fraction of remaining proteins with 20mM Imidazole. Sample 5: first elution, using 250mM imidazole. Sample 5: second elution, using 250mM Imidazole.
Using the gel run during characterization, the molecular weight of gbr22 in Sample 5 was estimated to be ~25 kDa. This was consistent with its known molecular weight of 25.79 kDa. Apart from gbr22, Sample 5 also had a very faint band of unknown protein at ~13 kDa, indicating an overall protein purity of ~95%. The heavy banding in Samples 3 and 4 showed that the buffer and wash were successful in removing impurities from supernatant Sample 2. Faint banding in Elution 2 indicated that Elution 1 was successful in outcompeting Ni-NTA. Large bands at ~13 kDa in Samples 1, 2, and 3 showed that some impurity was present in solution. This may have been introduced because of poor lab technique, or more likely overexpressed by the bacteria during growth phase. Poor loading of the gel led to bleeding of bands, and background stain could have been avoided by further washing of the gel.
Conclusion:
The methods used to overexpress, purify, and characterize a protein were addressed throughout this lab. E. coli BL21(DE3) was transformed with the genetic sequence for protein pGEM-gbr22 and made to overexpress the purple protein. Through the use of physical filters, chemical tags, and various buffers, the protein was purified and assessed for concentration level. In characterization, various samples of genetic material were run through a gel to conclusively verify that the protein being detected was actually gbr22. Being able to apply these same techniques in experiments involving proteins of interest can allow for experiments such as enzyme assays or other tests to test enzyme inhibition.
References:
[1] Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46.
[2] Mattaj, I. W. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/index.html (accessed April 16).
[3] Young, C. L.; Britton, Z. T.; Robinson, A. S., Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnol J 2012, 7 (5), 620-34.